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Monitoring enzyme catalysis with mass spectrometry

Academic Article
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Overview

authors

  • Bothner, B.
  • Chavez, R.
  • Wei, J.
  • Strupp, C.
  • Phung, Q.
  • Schneemann, Anette
  • Siuzdak, Gary

publication date

  • May 2000

journal

  • Journal of Biological Chemistry  Journal

abstract

  • Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition, k(cat) and K(m) values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics.

subject areas

  • Animals
  • Catalysis
  • Enzymes
  • Humans
  • Mass Spectrometry
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.275.18.13455

PubMed ID

  • 10788458
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Additional Document Info

start page

  • 13455

end page

  • 13459

volume

  • 275

issue

  • 18

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